From th,e Department of Biochrnistv, College of Medicine,. University of South Florida, Tampa, Florida 33612. The sigmoidal curves observed for rat ... Using radiation inactivation and immunoblotting tech- niques it was recently demonstrated ...
THEJOURNAL OF BIOLOGICAL CHEMISTRV
Communication
Vol. 260, No. 23, Issue of October 15,pp. 12391-12393,1985 0 1985 by The American Soclety of Bioloacal Chemlsts Inc.
Printed in U.’S.A.
Activation of Rat Liver Microsomal 3-Hydroxy-3methylglutaryl Coenzyme A Reductase byNADPH
EXPERIMENTAL PROCEDURES
EFFECTS OF DIETARY TREATMENTS* (Received for publication, June 24, 1985)
Gene C. Ness, SusanJ. Eales, Laura C. Pendleton, and MarthaSmith From th,e Department of Biochrnistv, College of Medicine, University of South Florida, Tampa, Florida 33612
The sigmoidal curves observed for rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase with NADPH as the varied substrate were markedly affected by feeding the animals diets containing colestipol, mevinolin and colestipolor cholesterol. Feeding of mevinolin and colestipol decreased the So.sfor NADPH from 270 to 40 PM, while cholesterol feeding increased the value to 1.3 mM. Immunoblotting analysis revealed that the M, 100,000form of HMG-CoA reductase predominated in cases where the So.5value was lowest, and the M, 200,000 species was the majorformwhere the So.5values were highest. Activation ofHMG-CoA reductase by NADPH was not dueto conversionofthe M, 200,000 formtothe 100,000 form.
Animals-Male Sprague-Dawley rats weighing 125-150 g were purchased from Harlan Industries of Madison, WI. The rats were housed under a 14-h light, 10-h dark cycle and were fed ad libitum Purina Rodent Laboratory Chow 5001, ground lab chow with 0.04% mevinolin and 2% colestipol, ground lab chow with colestipol only, or ground lab chow with 2% cholesterol, for 2 days. Colestipol is a bile sequestrant which actsto decrease the cholesterol feedback regulation of reductase by increasing conversion to bile acids, leading to a 3-$fold increase in activity. Mevinolin is a potent inhibitor of HMG-CoA reductase which acts to decrease effectivelyall metabolites derived from mevalonate, leading to a large compensatory increase in reductase protein. Given together, these drugs act synergistically to increase reductase activity 30-50-fold. All rats except the diurnal low group were killed at the 4thh of the dark period (diurnal high). The diurnal low group was killed at the 4thh of the light period. Livers were quickly removed, and lysosome-free microsomes were prepared in 0.25 M sucrose as previously described (3). HMG-CoA Reductase Assay-Activity was determined in microsomes containing intact reductase as a function of NADPH concentration using an NADPHgenerating system. Reaction mixtures contained potassium phosphate, pH 7.1, 100 mM; potassium chloride, 200 mM; glucose 6-phosphate, 30 mM; glucose-6-phosphate dehydrogenase, 1 unit; 60 p~ R,S-[’4C]HMG-CoA (specific activity 4400 cpm. nmol-l); and varying concentrations of NADPH ranging from 10 p~ to 20 mM. The mixtures were preincubated at 37 for 10 min in order to generate the NADPH. The reactions were started by the addition of microsomes. The final volume of the reaction mixture was 300 pl. In some experiments (Table 11),the microsomes were preincubated for 10 min, and thereactions were started by the addition of [14C]HMG-CoA.Incubation times ranged from 5 to 30 min. Reactions were terminated by the addition of 30 pl of2.4 N HC1. Incubation was continued for another 20 min to ensure lactonization of the [“C] mevalonate. The reactions were centrifuged to remove denatured protein. Then 100-pl aliquots were applied to 2.5-cm wide lanes on 0.76-mm thick Silica Gel G plates, and the mevalonolactone was separated using a solvent system of acetone:benzene (1:l). The area containing the mevalonolactone ( R F = 0.7) was scraped from the plate into a glass mini-counting vial. Four ml of ACS scintillation counting fluid was added. The amount of radioactivity present was determined by liquid scintillation counting. The nanomole of mevalonate formed was determined from the specific activity of the substrate determined under identical counting conditions and by correction for recovery using parallel reactions containing [3H]mevalonate. Protein concentrations of the microsomal suspensions were determined by a biuret method (4). Data were analyzed by V uersus S and Hill plots (log V/V,, - V versus log S ) . Maximum velocities were obtained from V uersus S plots employing sufficiently high concentrations of NADPH. Linear regression analysis was used to construct the best fit line to the data and thusdetermine the Hill coefficient ( n ~and ) theSo.sfor NADPH. Immunoblotting-The physical size of reductase present in microsomes was determined by immunoblotting analysis in the absence of B-mercaptoethanol as previously described?
“e
It has been demonstratedthat rat liver microsomal HMGCoAl reductase displays Sigmoidal kinetics when NADPH is the varied substrate (1). Atlow glutathione concentrations, less than 4 mM, a Hill coefficient of 2 wasobserved. This valueapproached 1 astheglutathioneconcentrationwas increased to 25 mM. ItappearsthatNADPHexerted this effect by allosteric interactionsrather than by donatingelectrons, as various structural analoguesof NADPH could substitute. The minimal structure required was that of 5’-AMP (2). Using radiation inactivation and immunoblotting techniques it was recently demonstrated that microsomal reductase exists in M,100,000 and 200,000 forms? The M , 200,000 form could be converted to 100,000 by isolating microsomes in media containing 10 mM dithiothreitol. The objectives ofthis study were to determine whether the observed NADPH-dependent allosteric kinetics of HMG-CoA reductaseare affected by various dietary treatments which RESULTS AND DISCUSSION are knownto markedly affect reductase activity, and whether an interconversion of the M, 100,000 and 200,000 forms might The effects of varying NADPH concentration on hepatic contribute to the allosteric kinetics. microsomal HMG-CoA reductase activity is shown in Fig. 1. Marked differences in the degree of sigmoidicity as a function * This work was supported inpart by United States Public Health with higher Service Grant HL18094. The costs of publication of this article were of dietarytreatmentwerenoted.Mkrosomes defrayed in part by the payment of page charges. This article must reductase activity required lower concentrations of NADPH therefore he hereby marked “advertisement” in accordance with 18 to achieve maximal activityand exhibited less sigmoidicity. U.S.C. Section 1734 solely to indicate this fact. TO definemorepreciselythechangesinthesigmoidal The abbreviations used are: HMG-CoA, 3-hydroxy-3-methylglu- curves caused by the various dietary factors, data were anataryl coenzyme A; So.5,substrate concentration required to achieve lyzed by Hill plots (Fig. 2). Table l presents a summary of 50% of maximal velocity. G. C. Ness, C. E. Sample, M. Smith,and M. J. McCreery, these data. The So.5 for NADPH progressively increases as manuscript submitted for publication. the specific activity decreases. These differences So.sinvalues
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of HMG-CoA Reductase
Regulation Allosteric
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TABLEI Effectsof dietary factorson kinetic parameters of microsomal HMGCoA reductase observed with NADPH as the varied substrate Reactions were initiated by the addition of microsomes without preincubation. Results are presented as means +- S.D. for three animals in each group except for the fasted condition. Dietary treatment
GSH Se.6 NADPH (5 DIM) trM
Mevinolin + colestipol Colestipol Diurnal high
0
I
I
I
I
0.5
1.o
1.5
2.0
[NADPH], mM FIG. 1. Effect of NADPH concentration on rat liver microsomal HMG-CoA reductase activity. Maximal reductase activity in terms of nmol.min-'. mg" and the milligrams of microsomal protein used in each reaction were 14.3 and 0.09, respectively, for mevinolin and colestipol-fed (A-A), 1.04 and 0.64 for colestipolfed ( C L - U ) , 0.29 and 0.64 for diurnal high (c".), 0.055 and and ), 0.020 and 1.23 for 24-h-fasted 0.64 for diurnal low (M ).-".( rats. Reactions contained 5 mM glutathione and were started by the addition of microsomes, Reductase activity is expressed as per cent of maximal for comparative purposes.
+ 0.6 W I
+ + -
Diurnal low
+-
Fasted 24 h
i-
Cholesterol-fed
+
-
-
33 f 5 39 f 7 165 f 7 164 f 7 234 f 74 271 2 149 396 f 50 514 f 147 1180 1100 2020 491 1340 f 716
Specific activity nmol. min" mg"
.
1.47 f 0.10 14.90 f 0.88 1.29 f 0.08 10.80 f 1.05 1.87 f 0.04 0.84 f 0.29 1.54 f 0.09 0.69 f 0.21 1.60 f 0.08 0.23 f 0.06 1.25 f 0.21 0.17 f 0.05 1.55 f 0.09 0.09 f 0.04 1.07 f 0.06 0.04 f 0.02 1.48 0.020 1.11 0.013 0.86 f 0.08 0.005 f 0.002 0.85 f 0.04 0.005 f 0.002
TABLEI1 Effect ofPreincubation on kinetic parameters of microsomal HMGCoA reductase observed with NADPH as the varied substrate Reaction mixtures were preincubated a t 37 "C for 10 min. Reactions were started by the addition of[14C]HMG-CoA. Results are presented as means f S.D. for three animals except for the cholesterol-fed condition.
SM
+
+ + + Diurnal high Diurnal low -+ Cholesterol-fed 3.58 + -
Mevinolin colestipol Colestipol
+ 1.0
+
-
Hill coefficient
nmnl .min" mg"
.
46 f 9 1.34 f 0.30 21.70 f 9.65 f 0.94 196 i: 1201.09 2 0.258.49 f 0.71 327 f 70 2.41 +- 0.371.46 957 f 328 1.40 f 0.320.95 f 0.22 1190 i:193 3.34 f 0.62 0.078 f 0.020 4260 & 1430 1.44 f 0.37 0.066 f 0.011 1410 f 2293.04 +- 0.54 0.036 f 0.022 4150 i:1020 1.41 f 0.40 0.033 f 0.018 2130 0.002 1.18 0.002 5820
x +0.2
E"
> >
-0.2
(3
9 - 0.6
-
1.0 J
1
1.0 -1.2 -1.6
0
1
I
-0.8
I
-0.4
1
0
L
+O(
LOG [NADPH] FIG. 2. Representative Hill plots ofHMG-CoA reductase activity. Liver microsomes were isolated from mevinolin and colescolestipol-fed (-), diurnal high (."-.), tipol-fed (A-A), diurnal low (o"--o) and 24-h-fasted (U rats. ) Reactions contained 5 mM glutathione and were started by the addition of microsomes.
might be due to different amounts of microsomal protein used in the assay. Thus assays with microsomes from colestipolfed, diurnal high, diurnal low, and 24-h fasted rats were also conducted a t identical protein concentrations. The same difference in So.&values was observed. It is of interest that thereported concentration of NADPH
in rat liver is about 500 FM ( 5 ) ,which lies in the midrange of the observed So.5values. Although the concentration of NADPH does not appear to change in response to fasting or feeding (5), NADPH could still affect the rateof the reductase reaction invivo owing to large changes in the So.5values (Table I). In contrast with the effects on So.5, the Hill coefficient, nH, was not affected as much by dietary manipulations. In most cases the nH was somewhat greater than 1 but always less than 2, indicating a degree of positive cooperativity. An exception was seen in microsomes from cholesterol-fed animals, where nH was consistently less than 1, suggesting negative cooperativity. Inclusion of glutathione in the assay always resulted in an increase in the Hill coefficient (Tables I and 11). When microsomes were preincubated with NADPH substantial differences in the sigmoidal curves were noted (Table 11). A 4-6-fold increase in the So.5values was seen. Inclusion of glutathione up to 25 mM reduced these values to near those seen without preincubation. This also resulted in Hill coefficients of 3 and greater with microsomes containing lower levels of reductase, i.e. diurnal high, diurnal low, and cholesterol-fed. Since, intracellularly, the reductase is exposed to both HMG-CoA and NADPH, it is felt that these values are not reflective of the in vivo situation. This experiment may,
Allosteric Regulation of HMG-CoA Reductase
F F -4824 DH C MC
0f
205
:f
97
66 FIG. 3. Immunoblotting of hepatic microsomal HMG-CoA reductase. Each lane contained 100 pg of microsomal protein from 48-h-fasted (F48); 24-h-fasted (F24); diurnal high (DH); colestipolfed (C); and mevinolin and colestipol-fed (MC) rats.The positions of prestained protein standards areindicated. Electrophoresis was conducted in the absence of P-mercaptoethanol.
NADPti
0
.05
.15
.30
3.0
?
0
r
X
205
E0
-
P
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microsomes with lower levels of reductase. Preincubation of reductase with NADPH has become standard practice largely as a resultof the observation that theproteolytically modified form of reductase is cold-liable and that NADPH could protect theenzyme (8).Intact native microsomal reductase is not cold-liable (9). To determine whetherthe observed differences in allosteric properties of microsomal reductase might relate to the portion of enzyme presentin the M , 100,000 and 200,000 forms, immunoblots in the absence of P-mercaptoethanol were performed (Fig. 3). The portion of enzyme present in the M , 100,000 form increases as the So, for NADPH decreases. In Fig. 3 equal amounts of microsomal protein were applied. Thus thelane containing proteinfrom rats fed mevinolin and colestipol is overloaded with respect to reductase immunoreactive enzyme. When less protein was applied, it was apparent that more than 80% of the reductase was present in the MI 100,000 species. It is striking that immunoreactive protein is not decreased even after 48 h of fasting when reductase activity was undetectable. The possibility that the allosteric activation of reductase by NADPH might result from conversion of the M, 200,000 form to theM, 100,000form was examined by immunoblotting in the absence of P-mercaptoethanol (Fig. 4). Samples were taken from reaction mixtures containing varying concentrations of NADPH. There was no evidence for formation of the M, 100,000 form even at NADPH concentrations where maximal activity was obtained. These data suggest that both forms might be capable of enzyme activity but have different requirements for NADPH. This finding is also consistent with NADPH acting at an allosteric site rather than providing electrons to effect the reduction of disulfide linkages in the M , 200,000 species. Thepresentdata suggest that dietary treatments profoundly affect the allosteric kinetics of HMG-CoA reductase, and that thisappears to be due to changes in theratio of the MI 200,000 species to the M , 100,000 species. How the interconversion of these two forms is accomplished in vivo in response to needs for mevalonate-derived products remains to be investigated. 1.
FIG. 4. Effect of NADPH concentration on immunoblots of HMG-CoA reductase from diurnal high rats. Reactions with the indicated millimolar concentrations of NADPH, 5 mM glutathione, and 60 p~ R,S-HMG-CoA were initiated by the addition of microsomes. Five min after starting the reactions, 100-pg aliquots were removed and mixed with 2 X Laemmli sample buffer. Electrophoresis was conducted in the absence of P-mercaptoethanol.
2.
however, suggest a possible role for HMG-CoA in protecting the reductase from NADPH-mediated inactivation. Comparing the results of Table I1 with those in Table I may provide some insight into certainreported discrepancies between changes in reductase activity and thosein cholesterogenesis (6,7). Often the observed change in reductase activity exceeded that of acetate or water incorporation into,cholesterol. This could be due to NADPH-mediated inhibition in
6.
3. 4.
5.
7. 8.
9.
REFERENCES Roitelman, J., and Schechter, I. (1984) J. Biol. Chem. 259,870877 Roitelman, J., and Schechter, I. (1984) Biochem. Biophys. Res. Commun. 125,902-907 Phillips, C.E., and Ness, G. C. (1984) Biochem. Biophys. Res. Commun. 119,772-778 Lee, Y. P., and Lardy, H. A. (1965) J . Biol. Chem. 240, 14271436 Passonneau, J. V., and Lowry, 0.H. (1974) in Methods of Enzymatic Analysis, (Bergmeyer, H. U.,ed) Vol. 4, pp. 2059-2066, Academic Press, New York Gibbons, G. F., Bjornsson, 0. G., and Pullinger, C. R. (1984) J . Biol. Chem. 259, 14399-14405 Bjornsson, 0.G., Pullinger, C. R., and Gibbons, G. F.(1985) Arch. Bwchem. Biophys. 238,135-145 Heller, R. A., and Gould, R. G. (1975) Biochim. Biophys. Acta 388,254-259 Ness, G. C., Degnan, M. G., and Smith, M. (1982) XZZ Znternational Congress of Biochemistry (Linnane, A. W., ed) p.119, International Union of Biochemistry, Perth
